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Home , Jonathon Pines, Spindle apparatus, Theodor Boveri, Walther Flemming

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The Role of Model Organisms in the History of Research

Mitsuhiro Yanagida

Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan Correspondence: [email protected]

Mitosis is a -cycle stage during which condensed migrate to the middle of the cell and segregate into two daughter nuclei before () with the aid of a dynamic mitotic spindle. The history of mitosis research is quite long, commencing well before the discovery of DNA as the repository of genetic information. However, great and rapid progress has been made since the introduction of recombinant DNA technology and discovery of universal cell-cycle control. A large number of conserved eukaryotic genes required for the progression from early to late mitotic stages have been discovered, confirm- ing that DNA replication and mitosis are the two main events in the cell-division cycle. In this article, a historical overview of mitosis is given, emphasizing the importance of diverse model organisms that have been used to solve fundamental questions about mitosis.

Onko Chisin—An attempt to discover new truths by checkpoint [SAC]), then (in which studying the past through scrutiny of the old. the chromosomes are aligned in the middle of cell), A (in which identical sister chro- matids comprising individual chromosomes LARGE SALAMANDER CHROMOSOMES separate and move toward opposite poles of ENABLED THE FIRST DESCRIPTION the cell), anaphase B (in which the spindle elon- OF MITOSIS gates as the chromosomes approach the poles), itosis means “thread” in Greek. In the and (the terminal phase of mitosis M19th century, pioneering researchers who during which chromosomes decondense, again developed light microscopic techniques dis- becoming invisible with light microscopy, the covered characteristic thread-like structures nuclear membrane reforms, and the spindle dis- in dye-stained cells before cell division. They assembles) before cytokinesis (cell division) named this stage “mitosis,” for the appearance (see Fig. 1 for terminology related to G1,G2, of the threads. The threads are now known to be S, and M phases, and Fig. 2 for a schematic of condensed chromosomes, which first become the progression of mitosis). visible with light microscopy during a mitotic In comparison with the whole-cell-division stage called . This is followed by pro- cycle, mitosis is a brief period during which metaphase (later known to be important as condensed chromosomes are accurately segre- this stage is controlled by the spindle assembly gated into daughter nuclei with the aid of an

Editors: Mitsuhiro Yanagida, Anthony A. Hyman, and Jonathon Pines Additional Perspectives on Mitosis available at www.cshperspectives.org Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a015768 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a015768

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M. Yanagida

Chromosome condensation segregation

M

Decondensation

G2 G1

S Sister chromatid cohesion

Chromosome DNA DNA replication

Figure 1. The consists of four phases: G1,S,G2, and M. Mitosis (M phase) is a brief period of the cell- division cycle. Blue denotes chromosomal DNA; red, centromere/. S phase, which comprises a period of DNA synthesis, is preceded by a gap (G1 means the first gap) in which there is no DNA synthesis. G1 phase is alternatively called the prereplicative phase. S phase is followed by another gap (G2). Again, there is no synthesis of DNA in G2 unless DNA damage must be repaired by replication. eventually enters M phase or mitosis, which completes one cell cycle. Morphological and biochemical events occurring in these phases are described. The cell-cycle concept was developed around 1953. The boundary between G2 and M is somewhat ambiguous because the initiation of prophase is difficult to define in some cells. Although the activation of -dependent kinase 1 (CDK1) kinase and two other protein kinases, polo and aurora B, are generally accepted as biochemical markers for the onset of mitosis, it should be noted that mitosis is a morphological (cell structural) event, and a number of visible cell structural mitotic markers have been proposed, such as nuclear membrane disassembly, chromosome condensation, spindle formation, kinetochore formation, etc. (see Fig. 2). The end of mitosis is also ambiguous. In telophase, chromosomes are fully segregated, but still condensed. Going into the G1 phase occurs after chromosome decondensation, reformation of the nuclear membrane around daughter nuclear , then followed by cytokinesis. In many cells, such as Physarum or vertebrate skeletal muscle cells, cytokinesis does not occur, producing multinucleate cells.

assemblage of pole-to-pole called research and also the founder of the spindle. In addition, there are short (see Fig. 3) (Paweletz 2001). Flemming de- microtubules that radiate from the spindle poles scribed the behavior of chromosomes during toward the cell cortex, and kinetochore micro- mitosis with amazing accuracy in an 1882 col- tubules that join the attachment region of chro- lection entitled, “Cell substance,nucleus and cell mosomes (called sister ). This is division.” For visualization of chromosomes, normally followed by a postmitotic event, cyto- Flemming used aniline dyes, which bind to kinesis, which generates two daughter cells. chromosomes. The first person to observe mitosis in detail Chromosome, in Greek, means colored was a German , Walther Flemming (“chroma”) body (“soma”). A chromosome is (1843–1905), who is the pioneer of mitosis an organized structure of DNA, protein, and

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Model Organisms in the History of Mitosis Research

Eukaryotic cells

Prophase

Chromosome

Kinetochore

Nuclear membrane Pole-to-pole microtubule Astral microtubule

Kinetochore microtubule

Metaphase

Anaphase A

Anaphase B

Telophase

Figure 2. Higher eukaryotic mitosis. In higher eukaryotic prophase, the nuclear membrane begins to degrade on the onset of chromosome condensation. In fungi, such as , the nuclear membrane remains during mitosis. (called “spindle pole bodies” in yeast) are duplicated and begin to form the mitotic spindle. In prometaphase, the full spindle forms and condensed chromosomes are attached to kinetochore microtubules. In metaphase, chromosomes are aligned at the middle. In anaphase A, sister chromatids are pulled toward opposite poles. In anaphase B, spindle extension occurs. Finally, in telophase, the nuclear membrane reforms.

RNA, which changes its form dramatically genetic material by Oswald Avery (Avery et al. during the cell cycle and under different phys- 1944). iological and physicochemical conditions. Sig- Flemming used a species of salamander as nificantly, most of Flemming’s work was per- the source of his material because salamanders formed before the rediscovery (1900) of the have very large chromosomes. The genome (a genetic principles discovered by haploid, or single set of chromosomes) size may (1822–1884). Flemming had no knowledge of be approximately 10 times larger than that of DNA, which was discovered as the “nuclein” humans, because it contains a large number of substance in 1869 by a Swiss biochemist, Fried- repetitious DNAs. In Flemming’s day, large cells rich Miescher, and much later identified as the containing large chromosomes were an obvious

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M. Yanagida

Figure 3. (A) Walther Flemming (image is part of the public domain and, therefore, is reprinted free from copyright restrictions). (B) (image is part of the public domain and, therefore, is reprinted free from copyright restrictions). (C) Barbara McClintock (image courtesy of the Barbara McClintock Papers, American Philosophical Society; copyright holder is unknown).

advantage. The large chromosomes of cells such materials. Chromosomes are gene transmitters. as thin epithelial cells of newt lungs have proven Third, there exists a relationship between the to be extremely useful in making high-resolu- location of individual genes on the chromo- tion movies of mitosis (Rieder and Hard 1990). some and frequencies of crossing over affecting Detailed behavior of individual chromosomes them. In addition, by observing aberrant mi- at the SAC (Li et al. 1993), which regulates toses, Boveri (2008) proposed that scrambled prometaphase, was clearly observed by Nomar- chromosomes produced by abnormal mitosis ski-differential interference contrast video mi- might be the cause of carcinogenesis, so that croscopy (Rieder and Alexander 1990). can originate from a single cell that di- vided abnormally. SEA URCHIN EMBRYOS REVEALED RAPID, REPEATED MITOSES MAIZE REVEALED CHROMOSOME PLASTICITY Theodor Boveri (1862–1915), a German biol- ogist, understood the importance of cell and BarbaraMcClintock (1902–1992),anAmerican chromosome research and he worked on the cytogeneticist, made several important dis- early development of sea urchins (Phylum Echi- coveries in and chromosome , nodermata). He discovered fast, repeated mi- using maize as her experimental organism. toses, revealing individual chromosomes and She mapped maize genes on chromosomes centrosomes at early embryonic stages. Because (McClintock 1929, 1931), discovered ring- embryonic sea urchin cells are highly transpar- shaped chromosomes (McClintock 1932), un- ent and divide every 20–30 min, they are ideal stable chromosome ends (McClintock 1941), for observing mitotic events in real time. Bo- and transposable genes (McClintock 1950). Al- veri’s fundamental contributions to the chro- though none of these was closely related to mosomal theory of inheritance, mitotic cell cy- mitosis, McClintock’s work made it possible to cle, and tumorigenesis are available in English understand certain behaviors of chromosomes, (Baltzer 1964; Boveri 2008). such as recombination and transposition. For Baltzer (1964) observed that Boveri’s work example, if telomeres of two chromosomes were established the foundation for the chromosome lost during cell division, the resulting daughter theory that identified chromosomes as the cells might produce ring-like chromosomes hereditary material. First, chromosomes are in- with two (special DNA regions dividual bodies in the . Second, dif- in which kinetochores attach to the spindle mi- ferent chromosomes carry different hereditary crotubules during metaphase). McClintock’s

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Model Organisms in the History of Mitosis Research

studies became very important when one con- Mazia and Dan (1952) developed cell-free siders the outcomes of unusual chromosome methods to isolate the mitotic apparatus from events, such as cases of unequal segregation or dividing sea urchin eggs (Strongylocentrotus transposition. In addition, she rightly pointed franciscanus and Strongylocentrotus purpuratus) out that differentiated cells often arise after in quantity. Their procedures were based on asymmetric mitosis. She stated that different selective solubilization of the cytoplasm sur- gene regulations arise from daughter cells rounding the . The isolated formed by asymmetric cell divisions in which structures at various stages in mitosis contained one daughter cell gained or lost something com- chromosomal and nonchromosomal parts and pared with the other (McClintock 1984). Stud- correspond well to the microscopic structures in ies of chromosome structure also flourished us- vivo. The isolated apparatus is birefringent, as ing plant cells because chromosomes of certain seen in polarized microscopy. The entire mitot- plant cells, such as lily, onion, and Haemanthus, ic apparatus behaved in the test tube as a single are very large. For example, meiotic behavior of physical entity. lily kinetochores could be observed in detail It was unclear whether the mitotic spindle with light microscopy (Matsuura 1951). How- and asters were required for cell division. Yukio ever, chromosome studies at the molecular level Hiramoto (1956) bravely removed the spindle using plants are relatively uncommon today. apparatus from dividing cells of sea urchin em- bryos using a micropipette. Cytokinesis (cell division) still occurred when the spindle was MITOTIC SPINDLE FOUND IN DIVERSE removed during anaphase or later, indicating that the spindle apparatus, although essential Polarization microscopy, using birefringence, for chromosomal movement, was not required was invented for observing the mitotic spindle for cell division per se. In some eggs, initiation in living cells, as spindle fibers connecting the of furrowing was even seen despite the removal spindle poles and chromosome are doubly of both spindle and asters during mitotic meta- refractive (Inoue and Dan 1951; Inoue 1953). phase. The position of the cleavage plane was Before observation of the mitotic spindle by not altered by elimination of the whole spindle polarization microscopy, the existence of the apparatus. Hiramoto concluded that furrowing “spindle fibers” was regarded with skepticism. results from an active function of the cell cortex. Because microtubules are dynamic epheme- It is now known that an actomyosin ring formed ral structures, fibrous mitotic elements were at the cell equator later in mitosis is essential for thought, bysome,to be afixation artifact. Shinya cell division (Mabuchi 1973). Inoue, who used polarization microscopy to ob- serve the mitotic spindle in living cells, resolved the dispute. The spindle was found in dividing CULTURED MAMMALIAN CELLS REVEALED NUCLEOCYTOPLASMIC INTERACTIONS cells of various organisms, including grasshop- IN MITOSIS pers, fruit flies, tube worm (Chaetopterus perga- mentaceous) oocytes, and pollen mother cells of Mammalian cell culture techniques have quite a Lilium longiflorum. Inoue and others also used long history, starting in the 19th century by a polarization optics to show the fragile of German zoologist, Wilhelm Roux. Cells isolated the spindle, which disappears after mitosis and from tissues were grown in primary cultures in the presence of microtubule-destroying poi- and then maintained as cell lines or stocked sons. Glutaraldehyde, a bifunctional crosslinker, for multiple use. Animal cell culture became a was later found to be a good fixative. Confirma- common technique in the 1970s. Rao and John- tion of the ubiquity of the mitotic spindle son performed key experiments that showed among eukaryotes, such as fungi, had to await the induction of mitosis by trans-acting (free- thin sectioning of cells for electron microscopy ly diffusing) factors (Johnson and Rao 1970; (Robinow and Marak 1966). Rao and Johnson 1970). Using the cell fusion

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M. Yanagida

technique, they mixed nuclei at different cell- yeast genetics paper, he isolated and character- cycle phases in the same cytoplasm (hetero- ized 400 temperature-sensitive (ts) mutants of karyon) and attempted to determine whether Saccharomyces cerevisiae, which were defective these nuclei at different cell-cycle phases could in certain aspects of the cell-division cycle influence one another. Their results were quite (Hartwell et al. 1970, 1971, 1973, 1974, 1991). striking. These ts mutants were unable to form col- When M-phase cells were mixed with G1,S, onies on rich media at 36˚C, but they grew nor- or G2 phase cells, premature (inappropriate) mally, or nearly so, at 23˚C. The mutants were mitosis occurred in the interphase nuclei, show- tested for loss of viability, change in morphol- ing that there were diffusible factors that could ogy, cell number increase, and the ability to promote M phase in the interphase nuclei. synthesize protein, ribonucleic acid (RNA), When S-phase nuclei were mixed with G1-phase and deoxyribonucleic acid (DNA) after a shift nuclei, G1 nuclei were induced to start S phase, from 23˚C (permissive) to 36˚C (restrictive suggesting that S-phase nuclei contain a diffu- temperature). Mutations were found that re- sible factor that induces DNA replication. When sulted in a preferential loss of the ability to per- S-phase nuclei were mixed with G2-phase nu- form protein synthesis, RNA synthesis, DNA clei, the G2 nucleus did not reinitiate S phase. synthesis, cell division, or cell-wall formation. G2-phase nuclei were refractory to the diffusible Time-lapse light microscopy was used to detect factor from S-phase nuclei. When G1-phase nu- ts mutants defective in gene functions needed at clei were mixed with G2-phase nuclei, no S or M specific stages of the cell-division cycle. phase occurred. These results strongly suggested By characterizing mutants of genes that that diffusible induction factors are produced control different stages of the cell cycle, these in the nuclei during S and M phases. The S- ts strains were called cdc (cell-division cycle) phase-promoting factor only works on G1 nu- mutants. For example, when cells carrying one clei. The M-phase-promoting factor works on cdc mutation arrest at a cell-cycle stage (the ex- everything. Rao and Johnson’s experiments us- ecution point), most cells end up with a tiny ing mammalian cells paved the way for under- bud that does not develop further. They are ar- standing mammalian cell-cycle regulations, and rested at bud emergence. When cells carrying later led to the discovery of maturation-pro- another cdc mutation terminate at mitosis, cells moting factor (MPF) and cyclin-dependent ki- display a large bud and are destined to arrest in nase (CDK). mid-nuclear division. Cells carrying another cdc mutation are defective in cell separation. They do not show a definite termination point be- YEAST cdc MUTANTS REVEALED GENETIC cause other processes of the cell cycle, such as CONTROL OF THE CELL CYCLE bud initiation and nuclear division, continue, For 3000–5000 years, mankind has depended despite the block in cell separation. on the budding yeast, Saccharomyces, for mak- After characterization of cdc mutants defec- ing bread and beer. In 1857, Louis Pasteur tive at different cell-cycle stages, particularly at (1822–1895) discovered the fermentation pro- initiation of DNA replication, bud emergence, cess using yeast. He showed that alcoholic fer- nuclear division (mitosis), and cell separation mentation was conducted by living yeast cells (cytokinesis), Hartwell et al. (1974) proposed a and not by a chemical catalyst. In the laboratory, model that accounted for the order of cell-cycle yeast has been intensively used by biochemists events that was deduced from the phenotypes to study enzymatic roles in various metabolic of budding yeast ts mutants. These pioneering pathways. Lehland Hartwell, a molecular biolo- genetic studies were performed before the age gist originally trained in mammalian DNA syn- of DNA cloning and sequencing and recombi- thesis, directed his attention to yeast genetics nant DNA technology. At the time of cdc mu- about 1965 and introduced a revolutionarily tant isolation, there was no concrete hope that new approach to cell-cycle research. In his initial genes responsive to mutations and molecular

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Model Organisms in the History of Mitosis Research

functions of gene products would be elucidated stages of maturation. The most effective cyto- in the near future. However, Hartwell and his plasm peaked in parallel with maturation. The colleagues identified CDC28 as the crucial cell- responsible substance was unidentified at the cycle regulator, which later turned out to be the time, but it was cytoplasmic, as the production catalytic subunit of CDK1, a fundamental cell- of MPF was not affected by removal of the nu- cycle regulator. cleus. MPF was later established to cause germi- The fission yeast, Schizosaccharomyces nal vesicle breakdown when injected into the pombe, is also an excellent model for studying frog (Xenopus oocytes) and to induce mitotic cell-cycle control, mitosis, and genome biology. metaphase in a cell-free system. S. pombe possesses approximately 5000 genes Seventeen years after the initial discovery, and is believed to have diverged from S. cerevi- Lohka et al. (1988) reported the purification siae about one billion years ago. Parallel studies of MPF from egg extract using ammonium are often useful because that which is true in sulfate precipitation and six chromatographic both often applies to vertebrates. Mitch- steps. Two protein bands were present in the ison and Leupold, respectively, initiated cell most highly purified preparations. This material physiology and genetics of S. pombe in the contained an intense protein kinase activity that 1950s (Mitchison 1957; Leupold 1958). S. phosphorylated . That same year, pombe vegetative cells are rod-shaped and the Maller and colleagues (Gautier et al. 1988) re- organism increases its length by growth. Using ported that one of the two components in the this property, Fantes and Nurse (1977) isolated purified MPF was actually the homolog of the cell-size mutants, later found to be defective fission yeast Cdc2 kinase (a homolog of budding in Cdc2, Wee1, and Cdc25, which are impor- yeast Cdc28). The other band was later identi- tant cell-cycle-regulating kinases and a phos- fied as mitotic cyclin (Hunt 2004), the level of phatase, respectively (Nurse 1990). yeast which underwent cell-cycle stage-specific deg- has only three chromosomes, and mitotic chro- radation by -mediated that mosome condensation is visualized using a led to the inactivation of the Cdc2–cyclin com- fluorescent probe, DAPI (1,4-diaminidino-2- plex. This finding resolved several enigmas re- phenylindole), for staining DNA (Toda et al. garding M-phase transitions in the cell cycle. 1981). The genome is an attractive system be- Exciting discoveries were reported almost si- cause it contains heterochromatin with histone multaneously from several laboratories, which H3 lysine-9 methylation in centromeres, telo- caused a coalescence of mitosis research that meres, and the mating-type locus and includes previously had consisted of people working in numerous noncoding RNAs (Bernard et al. disparate disciplines with different organisms, 2001; Volpe et al. 2002; Hirota et al. 2008). such as fission yeast, budding yeast, flies, clams, frog oocytes, and mammalian cells. The uni- fication of cell-cycle research thus occurred DISCOVERIES THAT MERGE MPF through the discovery of MPF and CDK as the WITH CDK conserved cell-cycle regulators from yeast to The Rao–Johnson studies showed that nucle- higher eukaryotes (Nurse 1990). Molecular bi- ar–cytoplasmic interactions are important for ology of cancer research was also enormously regulating mitosis. Masui and Markert (1971) influenced by the discovery of the basic mecha- published a paper entitled “Cytoplasmic con- nism of cell-division cycle control, symbolized trol of nuclear behavior during meiotic matu- by CDK. ration of frog oocytes.” They hypothesized the presence of a cytoplasmic factor called MPF in CYCLIN AND SECURIN DESTRUCTION the embryogenesis of frog (Rana pipiens) eggs IN YEAST AND MAMMALIAN MITOSIS treated with progesterone. They could identify cytoplasmic activities by injecting cytoplasm Using clam oocyte extracts, Hershko and col- from progesterone-treated oocytes at various leagues identified the large complex called a

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M. Yanagida

cyclosome (Sudakin et al. 1995) (alternatively For example, Hinnen et al. (1978) trans- called anaphase-promoting complex [APC]) formed a leucine-requiring yeast strain, leu2, (Peters et al. 1996; Hershko 1999) that contains using a plasmid that carried the yeast LEU2 cyclin-B-selective ubiquitin ligase activity. This gene. Resulting Leuþ transformants contained APC/cyclosome modifies mitotic cyclin by a plasmid carrying either the LEU2 gene or polyubiquitination, promoting its destruction chromosomally integrated gene dependent on by 26S proteasomes. The same complex was the presence or absence of the replication origin found to be necessary for ubiquitin-mediated in plasmid DNA. The plasmid DNA sequence degradation of fission yeast Cut2, budding yeast integrated into the yeast genomic DNA behaved Pds1, and human hPTTG1 (collectively called like the yeast genomic DNA in mitosis and mei- “securin”) that are essential for chromosome osis. A significant breakthrough was made when segregation (Funabiki et al. 1996; Yamamoto Clarke and Carbon (1980a,b) identified a func- et al. 1996; Jallepalli et al. 2001). (Securin/ tional yeast centromere. They first isolated the PTTG is not essential in the mouse; CDC10 gene, which is close to the centromere of can be regulated by Cdk phosphorylation.) Se- chromosome III, by transformation of ts cdc10 curin destruction is needed for proper chro- mutants isolated in the laboratory of Lehland mosome segregation and the signal sequence Hartwell. They were able to establish the direc- for destruction, called the destruction box, can tionality of a cloned piece of DNA with respect be swapped between fission yeast mitotic cy- to the genetic map. In the second stage of their clin Cdc13 and securin Cut2 so that the timing investigation, Clarke and Carbon successfully of destruction should be under the same signal isolated a short piece of a functional centro- pathway. The role of the APC/cyclosome is thus mere. When present on a plasmid carrying a to coordinate mitotic control with chromosome yeast chromosomal DNA replicator, this DNA segregation. The actual role of securin is to phys- (designated CEN3) enabled the plasmid to ically associate with a protease called separin/ function as a minichromosome, both mitoti- separase, essential for cleavage of the cally and meiotically. These circular minichro- subunit. Securin acts as a chaperone/inhibitor mosomes are stable in mitosis and segregate of separin/separase. The loss of securin pro- as yeast chromosomes in the first and second motes activation of separase activity. meiotic divisions. Indeed, the functional cen- tromere of budding yeast was rather short, only several hundred base pairs. In other organ- IDENTIFICATION OF CENTROMERES isms, such as fission yeast, flies, and mammals, IN BUDDING AND FISSION YEASTS however, the centromeres are far larger, requir- Eukaryotic gene cloning and sequencing studies ing different approaches for identification and first flourished using S. cerevisiae as the model isolation. because this organism contained a Analyses of artificially constructed mini- 2-mm plasmid that was exploited as a source chromosomes by pulsed-field gel electrophore- of extrachromosomal genes. Any gene inserted sis showed that the size of the S. pombe centro- into that plasmid (or integrated into the geno- mere is 30- to 130-kb long, much larger than mic DNA) with marker DNA could be iso- that of S. cerevisiae, which is on the order of lated and sequenced, leading to identification 0.1 kb (Chikashige et al. 1989; Hahnenberger of the gene product. Transformation, which et al. 1991; Takahashi et al. 1992). Linear mini- changes the properties of yeast cells by intro- chromosomes were obtained by double trun- ducing new or altered genes, was most powerful cation followed by the addition of telomeric for elucidating the properties of endogenous sequences. The circular minichromosomes genes. Basically, the same transformation meth- were isolated by the gap-repair method (Hahnen- od was used in many other organisms, including berger et al. 1989; Niwa et al. 1989). These 30- mammals, to identify and manipulate genes of to 160-kb minichromosomes were useful to interest. define the S. pombe functional centromere re-

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Model Organisms in the History of Mitosis Research

gions and also to determine the entire repeti- Szostak and Blackburn (1982) then shifted tious centromere sequence (Niwa et al. 1989; to the use of yeast and constructed a linear yeast Takahashi et al 1992). The pericentromeric re- plasmid by joining fragments from the termini peats are heterochromatic because histone H3 of Tetrahymena rDNA to a yeast vector. Thus, is methylated and heterochromatin protein 1 yeast can use DNA ends from distantly related (HP-1), which affects accurate chromosome organisms, suggesting that structural features segregation like Swi6, is abundant. The central required for telomere replication might be high- centromere region associates with histone H3- ly conserved in evolution. The linear plasmid like CENP-A and Mis12, which form the base was then used as a vector to clone telomeres of the kinetochore and are essential for equal from yeast. One Tetrahymena linear end was re- . Pericentromeric re- moved, and yeast fragments that functioned as peats are actually transcribed and directed by an end on a linear plasmid were selected. Szo- RNA interference, which flanks the central cen- stak and Blackburn (1982) successfully isolated tromere region. The central centromere region yeast telomeres and suggested that all yeast tethers CENP-A-like, Cnp1-containing nucleo- chromosomes appeared to have a common telo- somes and promotes kinetochore assembly in mere sequence. Yeast telomeres appear to be mitosis. A number of bound to central similar in structure to the rDNA of Tetrahyme- centromere regions are all conserved in higher na, regarding the presence of specific nicks or eukaryotes. Although the DNA sequence orga- gaps within a simple repeated sequence. nization of centromeres differs greatly among Telomeres are protected from fusion, degra- organisms, proteins bound to the centromere dation, or recombination, common properties and pericentromere regions are highly conserv- of DNA damaged by g-irradiation, which in- ed, allowing the conservation of segregation duces double-stranded breakage. An enigmatic mechanisms that require centromere-binding question was what kind of structure at the ends proteins. of linear DNA allows their complete replication. Shampay et al. (1984) showed that yeast chro- mosomal telomeres terminate in a DNA se- LINEAR DNA ENDS IN THE LARGE quence consisting of tandem irregular repeats NUCLEUS OF Tetrahymena HELPED of the general form C1-3A. The same repeat ISOLATE TELOMERES units could be added to the ends of Tetrahymena Ciliates are a group of protozoans character- telomeres, in an apparently non-template-di- ized by the presence of hair-like cilia. Unlike rected manner, during their replication on lin- most other eukaryotes, these organisms, such ear plasmids in yeast. as Tetrahymena thermophile, possess two dif- Greider and Blackburn (1985) then found a ferent nuclei. A micronucleus, containing ordi- novel activity in Tetrahymena cell-free extracts nary chromosomes, serves as the germ line that adds tandem TTGGGG repeats onto syn- nucleus, but does not express its genes, whereas thetic telomere primers. The single-stranded the large nucleus is generated from the micro- DNA oligonucleotides (TTGGGG)4 and TGT nucleus by amplification of gene-sized DNAs. GTGGGTGTGTGGGTGTGTGGG, containing Free ribosomal RNA genes were discovered by the Tetrahymena and yeast telomeric sequences, Gall (1974) in the macronucleus. Blackburn respectively, each functioned as primers for and Gall (1978) studied sequences occurring elongation, whereas nontelomeric DNA oligo- at their termini in the extrachromosomal re- mers did not. A novel telomere terminal trans- combinant DNA (rDNA) genes and found a ferase, later identified as the ribonucleoprotein tandemly repeated hexanucleotide C4A2 se- enzyme called telomerase, is involved in the ad- quence 50 (C-C-C-C-A-A)n 30, in which the pa- dition of telomeric repeats necessary for the rameter n ranges from 20 to 70. Thus, Tetrahy- replication of chromosome ends in eukaryotes. mena was an ideal organism for identification of Telomere shortening was later shown to be re- the linear DNA end sequence. lated to senescence in all eukaryotes.

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M. Yanagida

DIVERSE MITOTIC MUTANT PHENOTYPES II (Top2) mutation (Uemura and Yanagida BROADENED OUR UNDERSTANDING 1984). Centromere-associating protein mutants OF MITOSIS such as mis6 produced unequal chromosome Mutants defective in mitosis have been isolat- segregation, resulting in cells with large and ed from various model organisms from fungi small daughter nuclei (Hayashi et al. 2004). to mammals, with the aim of understanding Various aspects of chromosome segregation gene functions essential for mitosis. Mammali- could be understood through gene functions an culture cell ts mutants have also been isolated essential for mitosis. Some of them are closely (Nishimoto et al. 1978). Here, two model or- related to cell-cycle control, including stage-spe- ganisms, fission yeast and fruit flies, are consid- cific protein modification and proteolysis. SAC- ered as examples. Systematic screening of fission related APC/cyclosome ubiquitin ligase and yeast mitotic mutants (ts and cold-sensitive ubiquitin-mediated anaphase proteolysis lead [cs]) was performed. A number of genes were to the destruction of mitotic cyclin and securin, identified and their products were characterized whereas protein phosphatases (PP1, PP2A) and (reviewed in Yanagida 2005). Three principal protein kinases (PKA, aurora), other than CDK, chromosome segregation defects (arrest, cut, are required for controlling different stages of and unequal) were found for this organism at mitosis. Assembly and proper functioning of the restrictive temperature (Fig. 4). For exam- the mitotic kinetochore and spindle apparatus ple, b- nda3 cs mutant was mitotically are highly complex, requiring multiple gene arrested at 22˚C because of the absence of the functions. Key players in chromosome segrega- spindle, resulting in the activation of SAC (Hi- tion are cohesin, condensin, the securin–sepa- raoka et al. 1984), whereas DNA topoisomerase rase complex, Top2, and kinetochore microtu- II (top2) ts mutants displayed the drastic cut bule destabilizers. They function not only in phenotype. In these cells, cytokinesis bisected mitosis, but also in interphase displaying dis- the nucleus that had failed to divide dur- tinct functions. Most mitotic genes identified ing anaphase because of a DNA topoisomerase are conserved in higher eukaryotes, so that the basic mechanism of eukaryotic chromosome segregation in mitosis should, likewise, be large- ly conserved. There are some curious excep- tions, however. For example, fission yeast cen- Arrest tromere protein Mis18, required for priming centromeres to load CENP-A/cenH3 protein Cnp1, is conserved in vertebrates, but not in Cut phenotype nonvertebrates, such as fruit flies (Fujita et al. 2007). Concerning this essential centromere Unequal segregation protein, vertebrates are more similar to fungi. Wang’s discovery of DNA topoisomerase (Wang 1991) brought relief to many who worked on various aspects of DNA metab- olism, because it was apparent that DNA wind- ing, unwinding, catenation, and decatenation had to occur for DNA to be properly used and DAPI archived. However, no one had any idea how those functions were performed. The answer Figure 4. Three types of chromosome segregation was that many specific DNA topoisomerases defects in fission yeast mutants cultured at the re- strictive temperature. Arrest, b-tubulin nda3 mutant; control different aspects of the topological cut phenotype, top2 mutant; unequal segregation, problems of DNA (Wang 1991). To elucidate centromere-binding protein mis6 mutant. DNA is the role of topoisomerase in forming mitotic stained with DAPI. chromosomes, a biochemical genetic approach

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Model Organisms in the History of Mitosis Research

was used. S. pombe mutants defective in Top2 these kinases inaugurated a new mechanistic activity were isolated by assaying a great number approach to understand how the entire mitotic of mutant extracts. With ts and cs top2 mutants, process,including chromosome segregation and it was established that Top2 is required for both subsequent cytokinesis, is regulated. Although chromosome segregation and condensation CDK1 was inactivated during the transition (Uemura and Yanagida 1984; Uemura et al. from metaphase to anaphase, polo and aurora 1987). In the laboratories of Wang and Botstein, remain active until telophase because they also Top2 was shown to be essential for budding regulate cytokinesis. Drosophila bearing mutant yeast mitosis (Holm et al. 1986). Type 1 topo- forms of polo and aurora revealed severe defects isomerase partly overlaps in function (relaxing in mitosis and cytokinesis with pleiotropic de- activity) with Top2, so that the double mutant fects in chromosome segregation. Polo and au- produced a nonmitotic phenotype in which the rora are present from fungi to higher eukaryotes cell-cycle block occurred during interphase and (Lane and Nigg 1996). These are profoundly the nucleolus was destroyed (Uemura and Ya- important in orchestrating mitotic events, such nagida 1984). as CDK regulation, spindle formation, mainte- The curious cut (cell untimely torn) pheno- nance of centrosome structuralintegrity,centro- type, which bisected the nucleus during cytoki- some activation, SAC, chromosome cohesion nesis (Fig. 4), was used as a cytological marker and condensation, and progression of cytokine- to isolate other mutants with similar pheno- sis (Earnshaw and Cooke 1991; Golsteyn et al. types. A number of mutants producing cut- 1994; Kumagai and Dunphy 1996; Biggins and like phenotypes were isolated and their gene Murray 2001). products were identified. All cut-like mutants The concept of chromatid cohesion was ini- turned out to be defective in important mitotic tially developed in fruit fly genetics. In PubMed, steps, such as chromosome condensation, seg- the terminology of sister chromatid cohesion regation, activation of separase, control of ubiq- started with the papers of Orr-Weaver (Kerre- uitin-mediated proteolysis, spindle formation, brock et al. 1992; (Miyazaki and Orr-Weaver spindle elongation, cytokinesis, etc. (Yanagida 1994) that clarified the phenotype of the fruit 2005). Among them, mutants in top2, separase fly mei-S332 mutation that displayed a defect in cut1, and condensin cut3 produced highly sim- chromatid cohesion. Mei-S332/Shugoshin is ilar phenotypes. They may be implicated in the now known to protect cohesion at centromeres removal of interphase components from chro- in coordination with type 2A phosphatase (Ki- mosomes before chromosome segregation (Ya- tajima et al. 2004). The mutant mei-S332, which nagida 2009; Akai et al. 2011). Chromosome stands for “meiotic from Salaria 332” (isolated condensation may be actually visualized as a in Salaria, Rome, Italy), was originally described result of the removal of proteins and RNAs in 1968 (Sandler et al. 1968). The mutant from chromosomes before segregation. showed chromosome defects Drosophila is an attractive organism inwhich in homozygotes of both sexes, hinting that this to study both the rapid rounds of mitosis typical gene is required in a common meiotic process of and the longer cell for separating sister chromatids. A subsequent cycles of diploid tissues later in development paper (Goldstein 1980) suggested that mei-S332 (Glover 1989). Powerful molecular biological mutants are defective in sister chromatid cohe- studies of fruit fly mitosis became possible after siveness, which was felt to be an important fac- the discovery of Drosophila mutants and use of tor in chromosome segregation. Holloway et al. reagents, pioneered by Nuesslein-Volhard (St (1993) later investigated this issue using verte- Johnston and Nuesslein-Volhard 1992). Glover brate cells and showed that the inactivation of and associates discovered polo and aurora, pro- Cdk1 was not required for sister chromatid sep- tein kinases with important roles in the progres- aration, but proteolysis might be needed to dis- sion from early to late mitosis (Sunkel and Glov- solve the linkage (“glue”) between them. How- er 1988; Glover et al. 1995). The discovery of ever, their hypothesis that ubiquitin-mediated

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M. Yanagida

proteolysis was required turned out to be indi- sity and Okinawa Institute of Science and Tech- rect. There exists a protease that directly cleaves nology Graduate University (OIST) for lively the protein responsible for sister chromatid and stimulating discussions and a productive cohesion. Studies on budding yeast showed research environment. The present article is im- that the protein complex, called cohesin, was perfect in citing important discoveries and re- responsible for sister chromatid cohesion and search topics, as the field of mitosis is now so that cohesion was disrupted in mitosis (Guacci broad and deep. The author apologizes for the et al. 1997; Uhlmann et al. 1999, 2000). A thiol lack of citations of the work of many people who protease, separase, cleaves the cohesin subunit contributed significantly to the development of Scc1/Rad21, dissociating the ring-like cohesin the field. The author thanks Norihiko Nakazawa from chromosomes and allowing disjunction of for preparing illustrations in Figures 1 and 2. sister chromatids. The author is greatly indebted to Steven D. Aird (OIST) for editing the manuscript. FUTURE PROSPECTS FOR MITOSIS Although mitosis was first discovered and de- REFERENCES scribed in detail commencing in the 19th cen- Akai Y, Kurokawa Y, Nakazawa N, Tonami-Murakami Y, tury, molecular biological approaches assumed Suzuki Y, Yoshimura SH, Iwasaki H, Shiroiwa Y, Naka- importance only after the onset of recombinant mura T,Shibata E, et al. 2011. Opposing role of condensin hinge against replication protein A in mitosis and inter- DNA technology, that is, gene cloning and se- phase through promoting DNA annealing. Open Biol 1: quencing. Whereas a large number of essential 110023. proteins and their functional complexes during Avery OT, Macleod CM, McCarty M. 1944. Studies on the mitosis are now known and their number is still chemical nature of the substance inducing transforma- tion of pneumococcal types: Induction of transformation increasing, very few are understood mechanis- by a desoxyribonucleic acid fraction isolated from pneu- tically at the atomic level. In the near future, mococcus type III. J Exp Med 79: 137–158. biochemistry and structural biology may be- Baltzer F. 1964. Theodor Boveri. Science 144: 809–815. come the dominant methods for solving basic Bernard P, Maure JF, Partridge JF, Genier S, Javerzat JP, All- shire RC. 2001. Requirement of heterochromatin for co- questions in mitosis. hesion at centromeres. Science 294: 2539–2542. Thereafter, physiological, medical, and evo- Biggins S, Murray AW. 2001. The budding yeast pro- lutionary fields related to mitosis will flourish. tein kinase Ipl1/Aurora allows the absence of tension to For example, little is understood about the al- activate the spindle checkpoint. Genes Dev 15: 3118– 3129. teration of mitosis under various nutritional Blackburn EH, Gall JG. 1978. A tandemly repeated sequence and environmental stresses. Patterns of mitosis at the termini of the extrachromosomal ribosomal RNA may change greatly under different physiolo- genes in Tetrahymena. J Mol Biol 120: 33–53. gical, nutritional, and senescent conditions. Boveri T.2008. Concerning the origin of malignant tumours by Theodor Boveri. Translated and annotated by Henry Comparative studies of mitosis in stem and Harris. J Cell Sci 121: 1–84. nonstem cells are of interest, as mitosis is a plau- Chikashige Y, Kinoshita N, Nakaseko Y, Matsumoto T, Mu- sible stage for the origin of asymmetric proper- rakami S, Niwa O, Yanagida M. 1989. Composite motifs ties between daughter cells, which are essential and repeat symmetry in S. pombe centromeres: Direct analysis by integration of NotI restriction sites. Cell 57: features of cell differentiation. In the end, the 739–751. importance of evolutionary variations in mito- Clarke L, Carbon J. 1980a. Isolation of the centromere- sis must be stressed, as changes in mitosis are linked CDC10 gene by complementation in yeast. Proc known to be the rich resources of diversification Natl Acad Sci 77: 2173–2177. Clarke L, Carbon J. 1980b. Isolation of a yeast centromere of organisms. and construction of functional small circular chromo- somes. Nature 287: 504–509. Earnshaw WC, Cooke CA. 1991. Analysis of the distribution ACKNOWLEDGMENTS of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes The author is greatly indebted to past and pres- during metaphase and early events in cleavage furrow ent laboratory members in both Kyoto Univer- formation. J Cell Sci 98: 443–461.

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Model Organisms in the History of Mitosis Research

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The Role of Model Organisms in the History of Mitosis Research

Mitsuhiro Yanagida

Cold Spring Harb Perspect Biol 2014; doi: 10.1101/cshperspect.a015768

Subject Collection Mitosis

Emergent Properties of the Metaphase Spindle Chromosome Dynamics during Mitosis Simone Reber and Anthony A. Hyman Tatsuya Hirano Meiosis: An Overview of Key Differences from The Centromere: Epigenetic Control of Mitosis Chromosome Segregation during Mitosis Hiroyuki Ohkura Frederick G. Westhorpe and Aaron F. Straight Cytokinesis in Animal Cells The Biochemistry of Mitosis Pier Paolo D'Avino, Maria Grazia Giansanti and Samuel Wieser and Jonathon Pines Mark Petronczki The Centrosome and Its Duplication Cycle Aurea Mediocritas: The Importance of a Balanced Jingyan Fu, Iain M. Hagan and David M. Glover Genome Gianluca Varetti, David Pellman and David J. Gordon The Role of Model Organisms in the History of The Kinetochore Mitosis Research Iain M. Cheeseman Mitsuhiro Yanagida

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